Increased monomerization of mutant HSPB1 leads to protein hyperactivity in Charcot-Marie-Tooth neuropathy

Abstract

Small heat shock proteins are molecular chaperones capable of maintaining denatured proteins in a folding-competent state. We have previously shown that missense mutations in the small heat shock protein HSPB1 (HSP27) cause distal hereditary motor neuropathy and axonal Charcot-Marie-Tooth disease. Here we investigated the biochemical consequences of HSPB1 mutations that are known to cause peripheral neuropathy. In contrast to other chaperonopathies, our results revealed that particular HSPB1 mutations presented higher chaperone activity compared with wild type. Hyperactivation of HSPB1 was accompanied by a change from its wild-type dimeric state to a monomer without dissociation of the 24-meric state. Purification of protein complexes from wild-type and HSPB1 mutants showed that the hyperactive isoforms also presented enhanced binding to client proteins. Furthermore, we show that the wild-type HSPB1 protein undergoes monomerization during heat-shock activation, strongly suggesting that the monomer is the active form of the HSPB1 protein.

FIGURE 1.

Thermotolerance and chaperone activity of cell lines stably expressing CMT-causing HSPB1 mutations. A, to measure the HSPB1-mediated thermotolerance, cells stably expressing different HSPB1 isoforms were heat-shocked for 30, 60, and 90 min or left untreated. The cell number was determined 48 h after treatment using a fluorescence-based method. Cell survival was expressed by dividing the fluorescence signal from each time point by the respective non-heat-shocked control. Cells expressing HSPB1 mutations R127W and S135F presented higher thermotolerance than the cells expressing the wild-type protein, whereas cells expressing the P182L mutation were more sensitive. Results are mean values with ±S.D. error bars from two independent experiments with three repetitions each. B, experimental setup for the in vivo chaperone experiments. Cycloheximide was added to the cells before treatment to avoid translation of additional endogenous chaperones during the heat shock. Chaperone activity was calculated by dividing the luciferase activity from each condition to the respective non-heat-shocked control. C, in vivo chaperone activity for cells stably expressing different HSPB1 isoforms. Cells expressing HSPB1 mutations R127W, S135F, and R136W showed higher chaperone activity than cells expressing the wild-type protein. Results are mean values with ±S.D. error bars from three independent experiments with three repetitions each. Statistical differences to the wild-type HSPB1 stable cell line were tested using Student's t test and corrected for multiple comparisons using the FDR test (supplemental Fig. S2). *, p < 0.05; **, p < 0.01; ***, p < 0.001; HS-R0, heat shock and no recovery; HS-R180, heat shock and 180-min recovery; NHS, non-heat-shocked control. WT, wild type.

FIGURE 2.

Oligomeric state of CMT causing HSPB1 mutations. A, SEC profile from the wild type and R127W, S135F, and R136W mutants. Fractions were run on gel and subjected to Western blot using anti-V5 antibody. Black arrows and dotted lines, M_r as determined by a calibration run using proteins of known molecular size. The gray arrowheads indicate the strongest bands as determined by densitometry using ImageJ software. B, graphs showing the amount of the HSPB1 protein along the SEC fractionation and the elution peak shift for the R127W and S135F mutants. Signals for each fraction were normalized for the highest signal intensity and plotted as a smoothed curve. C, Western blot showing HSPB1 dimers. Western blots performed with non-reduced samples (top) permit the visualization of the three possible HSPB1 dimers: the dimer between two exogenous V5-tagged HSPB1 molecules (2× HSPB1-V5), the dimer between two endogenous HSPB1 molecules (2× HSPB1end), and the dimer between exogenous V5-tagged HSPB1 and endogenous HSPB1 (HSPB1end + HSPB1-V5). Note the higher monomerization for the R127W and S135F mutations. The R136W mutation presented a lower dimerization only with the endogenous protein. WT, wild type.

FIGURE 3.

Predicted structure of the HSPB1 α-crystallin domain dimer. HSPB1 dimer structure was predicted based on the crystal structure of the αB-crystallin α-crystallin domain (21). Each dimer subunit is represented in a different color (blue and green). A, predicted HSPB1 dimer structure with enlargement showing the wild-type position of the residues Arg-127, Ser-135, and Arg-136 (R127, S135, and R136), indicated by red arrows. The gray arrowheads show hydrogen bonds. B, the mutation R127W does not allow the formation of a hydrogen bond necessary for the stability of the β-sheet present in the dimer interface. C, the mutation S135F results in the projection of the phenylalanine aromatic R-group (shown in white) toward the Thr-139 residue from the opposite dimer subunit (shown in red), possibly avoiding the formation of the hydrogen bonds and destabilizing the dimer. D, in contrast, the mutation R136W does not seem to cause any obvious change in the dimer interface, possibly explaining the milder effects found for this mutation (residues Arg-136, Cys-137, and Phe-138 from the opposite dimer subunit are shown in red).

FIGURE 4.

Tandem affinity purification of protein complexes from cell lines stably expressing wild-type and CMT-causing HSPB1 mutations. Tandem affinity purifications were performed using cell extracts from HEK293 cells stably expressing CMT-causing HSPB1 mutations C-terminally fused to a double affinity tag (FLAG-TEV-Protein A). Eluates were run on a 10% NuPage gel and silver-stained for protein visualization. Note the clear general enhanced binding of the three hyperactive mutations R127W, S135F, and R136W. WT, wild type.

FIGURE 5.

Heat activation-induced monomerization of wild-type HSPB1. Cells expressing wild-type HSPB1 were heat-shocked for 30 min at 44 °C and allowed to recover for different time periods. A, Western blot (WB) showing the decrease in HSPB1 dimers after heat shock. Cell extracts under reducing and non-reducing conditions were run on gel and subjected to Western blot for HSPB1. B, the ratio between monomeric and dimeric bands for each condition was calculated from bands from three independent experiments. The increased monomerization of HSPB1 protein peaks at 30 min after heat shock. Results are mean values with ± S.E. error bars. Statistical differences to the NHS condition were tested using Student's t test. C, heat shock-induced phosphorylation of HSPB1. Using phospho-specific antibodies, we checked for the phosphorylation of HSPB1 dimers and monomers at different time points. Phosphorylated forms of HSPB1 were equally present in the dimeric and monomeric bands. D, the heat-induced HSPB1 monomerization is unaffected by its phosphorylation status. The heat-induced HSPB1 monomerization assay was repeated in the presence of the p38 inhibitor SB203580 or the vehicle (DMSO) control. Cells were pretreated with 20 μm SB203580 or DMSO for 2 h prior to heat shock. The inhibition of HSPB1 phosphorylation does not abolish its heat-induced monomerization behavior. Lower panels, control Western blots with HSPB1 phospho-specific antibodies showing the inhibition of HSPB1 phosphorylation by treatment with SB203580. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NHS, non-heat-shocked; HS-R0, heat shock and no recovery; HS-R30, heat shock and 30-min recovery; HS-R60, heat shock and 60-min recovery; P-S15, HSPB1 phosphoserine 15; P-S78, HSPB1 phosphoserine 78; P-S82, HSPB1 phosphoserine 82.